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Growth of neural precursor cells using umbilical cord blood serum and a process for the preparation for therapeutic purposes

Growth of neural precursor cells using umbilical cord blood serum and a process for the preparation for therapeutic purposes description/claims

This application is a Continuation in part of U.S. patent application Ser. No. 10/413,356, filed on Apr. 14, 2003, and also claims the priority from Indian Patent Application Serial No. 196212, filed on Apr. 16, 2003 in India.

The present invention provides for the use of sera separated from the clotted umbilical cord blood for growing human embryonic stem cells and adult cells such as Neural precursor cells, for therapeutic purposes in regenerative medicine. In particular, the present invention relates to transdifferentiation of CD+ 34, CD45− and CD133+ stem cells from Mononuclear cells derived from Umbilical Cord Blood to Neural precursors and such cells may be used in transplantation and repair of nerve damage, stroke, spinal injury, Parkinson's and Alzheimer's.

Stem cell technology is an emerging field that may yield many promising therapies. Stem cells are special cells that have the ability to develop into many different types of tissue, such as bone, muscle, nerve, etc. In theory, they could be grown into replacements for almost any part of the human body. Stem cells are typically found in the embryo and umbilical cord of an organism, and in reservoirs within the human body. Researchers hope that stem cells will provide a solution to cure diseases caused by cell failure, and for repairing tissues that do not repair themselves. Heart damage, spinal cord injuries, Parkinson's disease, leukemia, and diabetes are among diseases named in relation to stem cell research. Hence, researchers are of the opinion, that if these stem cells are controlled, they could cure a variety of debilitating diseases in the years to come.

Stem cells are separated into three distinct categories viz. Totipotent, Pluripotent, and Multipotent. Stem cells are best described in relation to normal human development. Thus, a fertilized egg is totipotent; a fertilized egg produces an entire organism. After several cycles of cell division, these totipotent cells begin to specialize, becoming pluripotent. As the embryo begins to develop, these pluripotent cells become multipotent, specifically producing blood, skin, nerve, or other types of body cells. Multipotent stem cells are envisioned to potentially treat a variety of muscular-skeletal and neural disorders. While stem cells are extraordinarily important in early human development, multipotent stem cells are also found in children and adults. For example, one of the best understood stem cells are the blood stem cells. Blood stem cells reside in the bone marrow of every child and adult, and in fact, they can be found in very small numbers circulating in the blood stream. Blood stem cells perform the critical role of continually replenishing the supply of blood cells—red blood cells, white blood cells, and platelets throughout the life span.

Stem cells are the building blocks of blood and immune systems. They form the white cells that fight infection, the red cells that carry oxygen and the platelets that promote clotting. Stem cells are normally found in bone marrow where they continue to generate new blood cells throughout the life span of an individual. The presence of these stem cells in the bone marrow has made marrow transplantation an important therapeutic modality in the treatment of a variety of malignant and non-malignant diseases. This is because of the realization that permanent clinical benefit from transfused blood cells can come from transplantation of multipotent haematopoietic stem cells. Besides bone marrow, Mobilized Peripheral Blood (MPB), and Umbilical Cord Blood (UCB) have also been used successfully for transplantation. In recent years although significant advances have been made in bone marrow transplantation (BMT), the basic problem of finding a suitable matching donor still remains. This is because a group of antigens expressed by the leukocytes called the human leukocyte antigens (HLA) need to match between the donor and the recipient. Further bone marrow harvesting is a painful and invasive procedure and many donors are unwilling to donate marrow. Therefore, the search for alternate sources of stem cells has led to the development of stem cell transplant protocols from different tissues like liver (Kochupillai 1991), mobilized peripheral blood (Benboubker 1995), and cord blood (Mayani 1998). Of these, cord blood has significant advantages over the others. Increasingly, experts say cord blood transplants have distinct advantages over more traditional bone marrow transplants in stimulating the growth of healthy white blood cells. Stem cells can be collected from the bone marrow. However, the collection procedure is invasive, time-consuming, requires an anaesthetic and is painful for the donor. Also, cord blood is easily available, involves a non-invasive collection procedure and is better tolerated in transplants across the HLA barrier.

Like bone marrow, umbilical cord blood is rich in stem cells. Umbilical cord blood is the blood that remains in the placenta and umbilical cord following birth. Until recently the placenta and umbilical cord were discarded after delivery as medical waste, but now research has shown that cord blood is a rich source of blood (haematopoetic) stem cells, which can be collected, processed and frozen for potential future use. An experimental procedure to use umbilical cord blood instead of bone marrow to treat immune diseases is gaining attention from doctors and patients.

Research in human developmental biology has led to the discovery of human stem cells (precursor cells that can give rise to multiple tissue types), including embryonic stem (ES) cells, embryonic germ (EG) cells, fetal stem cells, and adult stem cells. Recently, techniques have been developed for the in vitro culture of stem cells, providing unprecedented opportunities for studying and understanding human embryology. As a result, scientists can now carry out experiments aimed at determining the mechanisms underlying the conversion of a single, undifferentiated cell, the fertilized egg, into the different cells comprising the organs and tissues of the human body. Although it is impossible to predict the outcomes, scientists and the public will gain immense new knowledge in the biology of human development that will likely hold remarkable potential for therapies and cures.

Using cell replacement therapy, to cure diseases may prove to be one of the most significant advances in medicine. Unlike all current treatments that rely on surgical interventions or drugs that modulate cell activities, stem cells provide a replacement for dysfunctional or degenerating tissue.

Cell implantation offers hope for actually replacing nerve cells lost in Parkinson's and many other neurological diseases. Using cell replacement therapy, to cure diseases may prove to be one of the most significant advances in medicine. Unlike all current treatment that rely on surgical interventions or drugs that modulate cell activities, stem cells provide a replacement for dysfunctional or degenerating tissue. Clinical trials of fetal tissue transplantation, still underway, have developed methods for implanting cells into the brain and demonstrated the viability of the concept and promising results for at least some patients. One of the major problems in the cell transplantation is the need of large number of neuronal cells.

Cells are regarded as stem cells if they retain the capacity to renew themselves as well as more specialized progeny. Stem cells can be obtained from early embryo, fetal tissues, adult blood, and umbilical cord blood. Identification of the full term umbilical cord blood (which is discarded at birth), as a source has made haematopoietic stem cells more accessible for study and clinical use. Cord blood stem cells are multipotent. These stem cells in addition to the production of blood cells, have the ability to differentiate into cells of other tissue or organs. This ability has made cord blood stem cells more accessible for study and clinical use.

Umbilical cord blood is typically depleted of RBC and the leukocyte rich fraction is subjected to density gradient separation to yield mononuclear cell fraction (MNC).

Typically MNCs comprise hematopoietic and non hematopoietic cells. The hematopoeitc cells further comprises monocytes and lumphocytes.

Cord blood stem cells express are multipotent. These stem cells in addition to production of blood cells have the ability to differentiate into cells of other tissue or organs. This ability has made cord blood stem cells more accessible for study and clinical use. Umbilical cord blood is rich in many stem cells including hematopoietic precursors and therefore represents a good source of cells for hematopoietic reconstitution. However, little work has been done using stem cells or neuronal precursors found in umbilical cord blood for neural transplantation. Published U.S. Patent Application Serial No. 2002/0028510 A1 describes the use of cord blood mononuclear cells for treating neural disorders. In this patent publication, the cord blood mononuclear cells are differentiated into neuronal and glial cells using Fetal Bovine Serum (FBS). The scientists of the present invention have developed an improved method for growth and differentiation of the cord blood mononuclear cells. In the present invention, cord blood mononuclear cells are grown and differentiated into neural cells using human umbilical cord blood serum. Since the present invention uses human umbilical cord blood serum, the risks associated with the use of FBS are circumvented, thereby improving the engrafting ability of the cells.

Cord blood stem cells express CD34 antigen. CD 34 antigen has been commonly used as a marker for the enrichment and isolation of candidate stem cells. Cord blood stem cells can be isolated on the basis of the presence of this marker. CD34 positive cells can be isolated from mononuclear cells of cord blood. Cord blood mononuclear cells constitute about 1-2% of CD34 positive cells. On exposure to a novel environment, cord blood stem cells are known to transdifferentiate to various cells like neural cells, liver cells, bone, cartilage etc. The CD markers are used to typically select the required type of cells. For example CD 34 is a specific marker for hematopoietic stem cells, CD45 is a specific marker for lymphocytes.

Transdifferentiation is the ability of the adult stem cells from one tissue or organ, which can overcome their intrinsic restrictions upon exposure to novel environment perhaps via genomic reprogramming to cells of other organs either in vitro, or after transplantation in vivo.

Neural stem cell research is still in its early stages, is intriguing because scientists believe that the primiti Many neurological diseases such as Parkinson's disease, Alzheimer's disease, Multiple sclerosis, Huntington's disease, Amyotrophic lateral sclerosis and Cerebral ischemia including Stroke are characterized by degeration of neurons in the brain and spinal cord regions. Such neurological diseases result in the loss of neurons and these degenerated cells or neurons are not intrinsically replaced or repaired (During et al. 2001).

There is substantial evidence in both animal models and human patients that neural transplantation is a scientifically feasible and clinically promising approach to the treatment of neurological diseases and stroke, as well as for repair of traumatic injuries to brain and spinal cord. Nevertheless, alternative cell sources and novel strategies for differentiation are needed to circumvent the numerous ethical and technical constraints that now limit the widespread use of neural transplantation.

Neural stem cell research is still in its early stages, is intriguing because scientists believe that the primitive cells can transform into virtually any cell type in the body and could be a source of tissue or organs to cure diseases such as repair of nerve damage, strokes, spinal injury, Parkinson's and Alzheimer's. For years, researchers studying stem cells have been intrigued by the possibility that these cells might be useful to treat brain diseases. Recent studies have suggested neural stem cells transplanted into the brain can migrate throughout the brain and develop into other types of cells.

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